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The effects of the inoculum, pH, cation concentrations, and different lots of commercial media on the in vitro susceptibility of Clostridium difficile to fidaxomicin were examined. Of the factors evaluated, only pH alterations influenced the activity of fidaxomicin against C. difficile, noticeably reducing its activity at higher pH (≥7.9).
In recent years, the epidemiology of Clostridium difficile infections (CDIs) has been changing, and there are increasing numbers of CDI cases being reported each year (2, 3, 5, 7, 9, 12, 13, 15, 17, 19, 20, 21). While standard therapies for CDIs reduce the rates of morbidity and mortality, they are known to lead to high rates of recurrence, with 15% to 30% of patients demonstrating relapses in symptoms in the first few weeks after treatment is discontinued (9). Fidaxomicin (formerly known as OPT-80 and PAR-101) is a novel and narrow-spectrum macrocyclic compound (1, 6, 10) that is in clinical development for the treatment of CDIs. In a recent phase 3 trial, fidaxomicin-treated patients demonstrated better clinical outcomes; namely, they had significantly lower rates of recurrences than subjects who were treated with vancomycin (16).
The site of action of C. difficile is the large intestine, a milieu filled with vast numbers of different species of anaerobic flora that, through their metabolites, maintain a physiological pH ranging from 5.5 to 7 (11). Disruption of the gut flora by antibiotic therapy can therefore lead to pH changes, which can affect the pH-dependent activities of many antibiotics (4, 8, 21). Other environmental variables, such as divalent cation concentrations (including calcium and magnesium) and bacterial density, can also influence the antimicrobial activities of compounds. The dependence of the antibacterial activity on these factors is an important consideration, particularly for an unabsorbed antibiotic such as fidaxomicin that localizes and targets bacteria in the gut, where these parameters can vary greatly with diet and disease state.
The in vitro activities of antimicrobial compounds (expressed as the MICs) under conditions with such environmental variables are also important factors to be considered when a methodology for future in vitro testing is designed. Brucella agar, which is recommended by the Clinical and Laboratory Standards Institute (CLSI) (18) for use for MIC determination, is not standardized, and the consistency of the divalent cation concentrations has not been established. Moreover, the pH of the medium used under anaerobic conditions in a glove box may also vary with different gas mixtures, as the CO2 concentration in the gas mixture has the propensity to acidify the medium and can thus be a significant source of variability. Macrolides, as an example, show elevated MICs in the presence of CO2 (8). The inoculum size may also be difficult to standardize, given the variety of atmospheric conditions available for anaerobic susceptibility testing (H2/CO2 generator, evacuation/replacement method, or anaerobic chamber) and the duration of organism exposure to the aerobic atmosphere during benchtop manipulations.
In the study described here, we examined the anti-Clostridium difficile activity of fidaxomicin by comparing the MIC values obtained in the presence of different concentrations of divalent cations, pHs ranging from 6 to 8, inoculum density ranges of over 3 orders of magnitude, and various commercial lots of brucella broth.
C. difficile laboratory strains ATCC 9689, ATCC 700057, ATCC 43255, and ATCC 17857 and Eubacterium lentum laboratory strain ATCC 43055 were obtained from the American Type Culture Collection (ATCC); and the MIC values were determined by the CLSI agar dilution method (18). Broth microdilution is not a CLSI-validated method for testing the MICs of Clostridium; however, due to the potential inaccuracy of measuring the pH of solid agar after equilibration inside the anaerobic chamber, both broth and agar dilution methods were used and the results were compared for the assessment of the pH effects. With the exception of drug dilution, all MIC testing steps for both methods were performed inside a glove box under anaerobic conditions (10% H2, 5% CO2, 85% N2). Microtiter plates with diluted drugs were equilibrated for a minimum of 3 h inside the glove box, prior to addition of the inocula. All MIC testing runs were performed at least in duplicate. When the values for replicate runs varied, the mode was presented, or if the values for the replicate runs were evenly split between two values, the higher value was reported.
To evaluate the effect of the inoculum density on the susceptibility of C. difficile to fidaxomicin, a suspension of ~108 CFU/ml was prepared and serially diluted by 10-fold factors to obtain culture densities of 105 to 108 CFU/ml. The delivery of 2 μl to the agar medium yielded inoculum densities that ranged from 105 to 108 CFU/ml (102 to 105 CFU/spot). The fidaxomicin MIC for C. difficile strains ATCC 9689 and ATCC 700057 (MIC values, 0.063 and 0.125 μg/ml, respectively) remained the same at all the concentrations tested, whereas the vancomycin MICs increased progressively with increasing inoculum concentrations, with the highest inoculum density showing a 4-fold rise in MIC over that obtained with the lowest inoculum density. Similarly, reproducible MIC values with other C. difficile strains have been observed in our laboratory (data not shown) at different inoculum concentrations.
The concentrations of the calcium and magnesium divalent cations in commercial brucella agar were determined by Laboratory Specialists, Inc., to be 21 and 33 mg/liter, respectively. Additional amounts of divalent cations were added (in the form of either calcium chloride or magnesium chloride) to obtain media with calcium ion concentrations of 21, 30, and 57 mg/liter or magnesium ion concentrations of 33, 45, and 75 mg/liter. The fidaxomicin MIC values for C. difficile ATCC 9689 and ATTC 700057 remained identical (0.063 and 0.125 μg/ml, respectively) for all cation concentrations tested (Table (Table1).1). Quality control susceptibility testing was conducted with vancomycin in medium with unaltered cation levels and consistently produced the expected MIC value of 1 μg/ml.
Three different lots of supplemented brucella agar medium were used on three separate days to compare the reproducibility of the fidaxomicin MIC values with each lot for three C. difficile strains (ATCC 9689, ATCC 43255, and ATCC 17857). The MIC assays were controlled by testing the activity of clindamycin against the quality control organism, Eubacterium lentum. As an internal control, the activity of metronidazole against C. difficile, which in our laboratory has been about 0.25 to 0.5 μg/ml, was monitored. The fidaxomicin MICs were unaffected by the different medium lots and remained within one 2-fold dilution of each other (Table (Table22).
The susceptibility of C. difficile (ATCC 9689 and ATCC 700057) to fidaxomicin was evaluated over a pH range of 6 to 8 by both the CLSI agar and the CLSI broth microdilution methods. With the agar dilution method, the fidaxomicin MIC was determined over a pH range of 6.2 to 8.0. In order to achieve the desired anaerobic pH for susceptibility testing, two different buffers [100 mM NaH2PO4 and N-Tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid (TAPS)] were added to the media at pH 7 and 8, respectively. To compensate for the reduction of the pH in the medium inside the anaerobic glove box (which occurred even with buffered media), the titer in the medium in ambient air, which was above the desired anaerobic pH, was first determined. Following equilibration inside the chamber with 5% CO2 for 3 h, the anaerobic pH was verified with a portable pH meter with a flat-bottomed pH probe. Vancomycin, used as a control, was tested only at about pH 7. The results showed no increase in the fidaxomicin MIC between pH 6.2 and pH 7; however, the fidaxomicin MIC increased by 8-fold with treatment at the highest pH (pH 7.9). This increase in the MIC was verified by repeating the MIC run at the highest pH (pH 8.0).
The fidaxomicin MICs, obtained by the agar dilution method, were verified by the broth microdilution method over a pH range of 6 to 8. Since the pH of the unbuffered brucella broth, the titer of which was determined in ambient air over a pH range of 5 to 9, dropped significantly over a pH range of 5 to 7.5 inside the glove box environment, buffer was added in the subsequent experiments to resist the pH shifts caused by anaerobic equilibration. Addition of buffer either as 10 mM or 100 mM (NaH2PO4·H2O, pH 7.0; morpholinepropanesulfonic acid, pH 8.0; or TAPS, pH 9.0 [the pH values are those in ambient air]) to the broth medium (with a pH above 6) resulted in final anaerobic pH ranges of from 6 to 7.6 and 6 to 8.1, respectively. Data from all experiments verified that the MIC values for both fidaxomicin and vancomycin increased with increasing pH. While the organism grew poorly at pH 5.0, at pHs above 6.5, the log of the MIC values for both drugs increased in a roughly linear fashion with the increase in the pH; the MIC values for both drugs at pH 7.5 and pH 8.1 were 8- to 16-fold those obtained by treatment at the lowest pH, pH 6 (Fig. (Fig.11).
Overall, with both methods of susceptibility testing and across various concentrations of buffer salts, the fidaxomicin and vancomycin MIC values increased with increasing pH. The high MIC values at basic pH, which has also been reported for other drugs (4, 21), may be due to increasing deprotonation of the phenolic hydroxyl groups of both compounds, forming a charged species that is expected to be less able to permeate bacterial cells. In contrast, under more acidic conditions, the antibiotics will be mostly protonated and should thus permeate the cell membrane more efficiently. It is unlikely that MIC trends are the result of the effect of pH on the organism density, as the level of growth (monitored by visual examination) did not always rise with increasing pH. Similarly, it is unlikely that MIC trends are the result of the interaction of the drug with buffer at the high concentrations used. Additional experimentation with various concentrations of buffers (at physiological pH) by the checkerboard method (against aerobic organisms, to avoid the shift in the pH from that in the atmosphere of the glove box with a high CO2 concentration) demonstrated no buffer effect on the drug MIC (data not shown).
In summary, the activity of fidaxomicin against C. difficile was unaffected by the inoculum concentrations, cation concentrations, or commercial lot of medium used; and the drug remained active at physiological pH, indicating that fidaxomicin in the setting of CDIs would be expected to have full and unaltered antimicrobial activity under the physiological conditions in the human intestine.
Published ahead of print on 22 March 2010.